ORIGINAL RESEARCHpublished: 18 July 2017

doi: 10.3389/fmicb.2017.01319

Frontiers in Microbiology | www.frontiersin.org 1 July 2017 | Volume 8 | Article 1319

Edited by:

Oscar Daniel Salomón,

National Institute of Tropical Medicine,

Argentina

Reviewed by:

Lorenzo Zammarchi,

University of Florence, Italy

Amer Hayat Khan,

Universiti Sains Malaysia, Malaysia

*Correspondence:

Joan-Pau Millet

jmillet@aspb.cat;

juampablomillet@gmail.com

Specialty section:

This article was submitted to

Infectious Diseases,

a section of the journal

Frontiers in Microbiology

Received: 13 January 2017

Accepted: 29 June 2017

Published: 18 July 2017

Citation:

Millet J-P, Montalvo T, Bueno-Marí R,

Romero-Tamarit A, Prats-Uribe A,

Fernández L, Camprubí E, del Baño L,

Peracho V, Figuerola J, Sulleiro E,

Martínez MJ, Caylà JA and Zika

Working Group in Barcelona (2017)

Imported Zika Virus in a European

City: How to Prevent Local

Transmission?

Front. Microbiol. 8:1319.

doi: 10.3389/fmicb.2017.01319

Imported Zika Virus in a EuropeanCity: How to Prevent LocalTransmission?Joan-Pau Millet 1, 2*, Tomàs Montalvo 2, 3, Ruben Bueno-Marí 4, Arancha Romero-Tamarit 1,

Albert Prats-Uribe 1, 5, Lidia Fernández 3, Esteve Camprubí 1, Lucía del Baño 1,

Victor Peracho 3, Jordi Figuerola 2, 6, Elena Sulleiro 7, Miguel J. Martínez 8, 9, Joan A. Caylà 1, 2

and Zika Working Group in Barcelona

1 Servicio de Epidemiología, Agència de Salut Publica de Barcelona, Barcelona, Spain, 2CIBER de Epidemiología y Salud

Pública, Barcelona, Spain, 3 Servicio de Vigilancia y Control de Plagas Urbanas, Agencia de Salud Pública de Barcelona,

Barcelona, Spain, 4 Laboratorios Lokímica, Departamento de Investigación y Desarrollo (I+D), Valencia, Spain, 5Unitat

Docent de Medicina Preventiva i Salut Pública Parc Salut Mar-Universitat Pompeu Fabra-Agència de Salut Pública de

Barcelona, Barcelona, Spain, 6 Estación Biológica de Doñana, Consejo Superior de Investigaciones Científicas, Sevilla, Spain,7Microbiology Department, Hospital Vall d’ Hebron, PROSICS Barcelona, Universitat Autònoma de Barcelona, Barcelona,

Spain, 8Department of Microbiology, Hospital Clinic of Barcelona, Universitat de Barcelona, Barcelona, Spain, 9 ISGlobal,

Barcelona Centre for International Health Research (CRESIB), Hospital Clinic of Barcelona, Universitat de Barcelona,

Barcelona, Spain

Background: On February 1st 2016 the WHO declared the Zika Virus (ZIKV) infection

a worldwide public health emergency because of its rapid expansion and severe

complications, such as Guillain-Barré Syndrome or microcephaly in newborn. The huge

amount of people traveling to endemic areas and the presence of Aedes albopictus in

Barcelona increase the risk of autochtonous transmission. The objective of this study

was to describe the first ZIKV cases diagnosed in our city and to analyze the surveillance,

prevention, and control measures implemented to avoid autochthonous transmission.

Methods: An observational cross-sectional population-based study in Barcelona,

Spain was performed.An analysis of the socio-demographic, epidemiological, clinical

characteristics, and mosquito control activities of the ZIKV cases detected between

January 1st and December 2016 was carried out using a specific ZIKV epidemiological

survey of the Barcelona Public Health Agency.

Results: A total of 118 notifications of possible ZIKV infections were received,

and 44 corresponded to confirmed cases in Barcelona residents.Amongst these,

the median age was 35 years and 57% were women. All cases were imported,

48% were Spanish-born and 52% foreign-born. Dominican Republic was the most

visited country amongst foreign-born patients and Nicaragua amongst Spanish-born.

The most frequent symptoms were exanthema, fever, and arthralgia. Among the 24

diagnosed women, 6 (25%) were pregnant. There was one case of microcephaly

outside Barcelona city. Entomological inspections were done at the homes of

19 cases (43.2% of the total) and in 34 (77.3%) public spaces. Vector activity

was found in one case of the 44 confirmed cases, and 134 surveillance and

vector control were carried out associated to imported ZIKV cases. In all cases

prevention measures were recommended to avoid mosquito bites on infected cases.

Millet et al. Zika Surveillance to Prevent Local Transmission

Conclusion: Epidemiological and entomological surveillance are essential for the

prevention of autochthonous transmission of arbovirosis that may have a great impact

on Public Health.The good coordination between epidemiologists, entomologists,

microbiologists, and clinicians is a priority in a touristic city with an intense relationship

with endemic countries to minimize the risk of local transmission by competent vectors.

Keywords: arbovirus, epidemiology, global health, Guillain-Barré syndrome, microcephaly, public health,

mosquito, Zika virus

INTRODUCTION

Arboviruses are a group of viruses transmitted througharthropods and many of them are capable of producing infectionin humans. West Nile virus (WNV), Chikungunya (CHIKV),dengue (DENV), or Zika virus (ZIKV) are emerging arboviruseswith a high potential of generating epidemic outbreaks (Whiteet al., 2016). ZIKVwas discovered in 1947 in Uganda by scientiststhat were performing a surveillance of the yellow fever virus ina forest called Zika (Hayes, 2009; Bulletin of the World HealthOrganization, 2016). This virus is transmitted by mosquitoes andAedes aegypti is the main known vector (Ayres, 2016; Chouin-Carneiro et al., 2016). Recent infectivity studies in laboratoryconditions revealed that Aedes albopictus is also susceptible toZIKV virus infection, since the virus is replicated, disseminatedand can reach to salivary glands. However, the efficiency ofthis infective process is lower in comparison with A. aegypti,which clearly shows the highest ZIKV vector competence amongmosquitoes (Chouin-Carneiro et al., 2016; Di Luca et al., 2016;Jupille et al., 2016). Moreover, it is important to note that A.albopictus has been found infected also in wild populations inendemic areas as Gabon, in Central Africa (Grard et al., 2014).

The first cases of infection by ZIKV in humans werediagnosed in Uganda and Nigeria in 1952 (Macnamara, 1954).Throughout the second half of 20th century, the ZIKV hadexpanded to countries in Africa and Asia; India, Egypt, Malaysia,Mozambique, Nigeria, the Philippines, and Vietnam. Until 2007,only 16 cases in humans had been reported. It is at this time,when the ZIKV expands to the Yap Island, in the Pacific(Federal State of Micronesia), where the first great outbreakwas reported (Duffy et al., 2009). Other large outbreaks werereported in the French Polynesia (2013–2014) and in Brazil in2015, where the first ZIKV autochthonous case in Latin Americawas reported. This outbreak has been the origin of the PublicHealth crisis initiated in 2016. By the starts of February 2016,local transmission of ZIKV had been reported frommore than 20countries and territories in the Americas (Bulletin of the WorldHealth Organization, 2016).

On February 1st 2016 theWorld Health Organization (WHO)declared the ZIKV epidemic a worldwide health emergency. Thistook place after the appearance of three epidemiological alerts

Abbreviations: CHIKV, Chikugunya; DENV, Dengue; GBS, Guillain-BarréSyndrome; PHAB, Public Health Agency of Barcelona; STD, Sexual TransmittedDisease; SUPCS, Surveillance and Urban Plague Control Service; WHO, WorldHealth Organization; ZIKV, Zika Virus.

related to outbreaks of congenital microcephaly and Guillan-Barré Syndrome (GBS) cases due to ZIKV in countries suchas Brazil, France, USA, and El Salvador (U. S. Department ofHealth and Human Services, et al., 2016). Although a decline inZIKV infections has been reported in some countries, or in someregions of countries, surveillance needs to remain high (WHO,2016a).

Within Europe, Spain is one of the countries with highrisk of autochthonous cases of ZIKV infection (WHO, 2016b),due to the presence of A. albopictus (Grard et al., 2014;Bueno, 2016) (commonly named “tiger mosquito”) in variousregions and due to the great cultural, commercial, touristic,and migratory relationship with Latin America (Díaz-Menéndezet al., 2016). Consequently in April 2016 a National Plan ofpreparedness and response against DENV, CHIKV, and ZIKVwas established. Its objective was to reduce the impact and therisk of establishment of these emerging diseases in Spain (MSSSI,2016).

In Spain, the first imported case of ZIKV was detected inDecember 2015–January 2016 (Bachiller-Luque et al., 2016).In Barcelona, both imported ZIKV cases and A. albopictus (acompetent vector for ZIKV) overlap in space and time. With theobjective of controlling the imported ZIKV cases and preventautochthonous cases, the public health services of the city werereorganized. This rearrangement was included within a moreglobal Surveillance and Control of Arbovirosis Program that wasalready in place since 2014 that includes other emerging virusestransmitted by A. albopictus such as DENV, WNV, and CHIKV(Agència de Salut Pública de Catalunya, 2015; González et al.,2017).

The objectives of this paper were to describe theepidemiological and entomological surveillance for ZIKVcases in Barcelona (Spain) and to describe the improvements inthe procedures to control ZIKV and other emerging arbovirosisto reduce the risk of an autochthonous outbreak.

METHODS

DesignA descriptive observational cross sectional population-basedstudy was performed in the city of Barcelona.

Study Period and PopulationThe notified and confirmed ZIKV cases among residentsin Barcelona city from January 1st to December 31st 2016were studied. All cases were detected and followed by theEpidemiology Service of the Public Health Agency of Barcelona

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Millet et al. Zika Surveillance to Prevent Local Transmission

(PHAB) through the notifiable disease register. Entomologicalsurveillance period starts on March and finishes on Novemberbecause the low temperatures prevent vector activity. Thecases that did not reside in Barcelona and those that werenot confirmed by the laboratory were excluded from theanalyses.

ZIKV, CHIKV, and DENV are diseases of mandatorynotification in Spain. In Barcelona an active epidemiologicalsurveillance system is carried out based on a closecommunication among doctors, laboratories and the publichealth nurses from the Epidemiology Service. The circuit andprocedures followed from the notification of a possible case untilentomological inspections are listed in Figure 1.

Definitions and Case ClassificationThe case definition used at the PHAB is described in the“National Plan for preparation and response against vectortransmitted diseases” and in the “Protocol for surveillanceand Control of mosquito transmitted arbovirosis in Catalonia”updated on 14 June 2016. The cases were classified according toclinical, epidemiological and laboratory criteria in four categories

(Díaz-Menéndez et al., 2016; González et al., 2017).

1. Probable case: person that complies with the clinical criteriawith or without epidemiological criteria and that complieswith the laboratory criteria of probable case.

2. Confirmed case: person that complies with the clinical criteriawith or without epidemiological criteria and that complieswith the laboratory criteria of confirmed case.

3. Imported case: when the beginning of the symptomsoccur until 15 days after abandoning a ZIKV epidemicarea.

4. Autochthonous case: if there is no record/history of trip toendemic area within the last 15 days previous to the beginningof symptoms (Agència de Salut Pública de Catalunya, 2015).

The days of viremic phase in Barcelona since the arrival date werecalculated for each patient. The viremic period was estimatedfrom the natural history of the infection reported in the literature.For the viremic period, we assumed 7 days starting from the dayof symptoms onset. However, we also simulate how many peoplewould be viremic in Barcelona with different theoric values: 8 and9 days of viremic period.

FIGURE 1 | Coordination diagram from notification of a case of ZIKV infection to the intervention of the entomologist in the city of Barcelona.

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Millet et al. Zika Surveillance to Prevent Local Transmission

Variables and Source of InformationThe different epidemiological variables were collected fromthe epidemiological survey of Catalunya specific to ZIKVinfection. Socio-demographic variables (age, sex, country oforigin, neighborhood/district of residence), clinical variables(date of beginning of symptoms, viremic period, fever, asthenia,artharlgia, arthritis, conjuntival hyperemia, cephalea, no-purulent conjunctivitis, exanthema, myalgia, complications,hospitalization), diagnostic variables [date of diagnosis,laboratory results (IgG, IgM, PCR)], and epidemiologicalvariables (way of transmission, pregnancy, and gestation weekif affirmative, country or countries visited, dates of departure,and arrival to Barcelona, cause of trip, number of bites, place inwhich bites where received, mobility of the cases and if activitiesof prevention, confinement, or personal protection were given)were collected. The median time elapsed (in days) between thesymptoms onset of patients and the first medical consultation,the notification and the laboratory confirmation, and theimplementation of vector control activities were calculated.

The Surveillance and Urban Plague Control Service(SUPCS) at PHAB recorded different information duringtheir entomological inspections related to the cases: placesvisited during the viremic period, vector activity at caseresidency, capture of adult vectors with BG traps, detection ofmosquito breeding sites, and presence of virus in the vector.

Surveillance Procedures and VectorControlIn order to minimize the risk of ZIKV transmission throughlocal mosquito bites during the viremic period, preventionmeasures where recommended by the Epidemiology Service.These measures included personal protection measures againstmosquito bites (type of clothing, use of mosquito repellent,etc.), and house confinement during the viremic period andrecommendations regarding safe sex.

Based on the collected information in the epidemiologicalsurvey, the SUPCS carried out an entomological inspection inat least two places: the public space around the case’s place ofresidence and in the patient’s own home. All the risk areas forthe proliferation of mosquitoes were included in a GeographicalInformation System (GIS) to speed up the inspections. Gutters,fences, ornamental fountains and small artificial containers,within a buffer of 150m in relation to the case’s place of residence(habitual flying ratio of A. albopictus in the urban setting) wereincluded in the GIS.

In our study, informative notes with recommendations toprevent the proliferation of larvae breeding sites in the homewere distributed during the inspections of more vulnerableresidential areas close to the imported case. The entomologicalinspections in private homes were analogous to the alreadydescribed procedures for public areas. The areas were inspectedfor detecting larvae breeding spot sites and adult mosquitoes.It’s worth mentioning that these activities are essential in thereduction of the risk of disease expansion because the contactbetween vectors and the infected host is likely to occur at home.The need to obtain an authorization from the patient to be able

to carry out the inspection was an important limiting factor incomparison with the interventions in public spaces.

The collected entomological material was examined andidentified and females were analyzed in pools for the detectionof virus presence by RT-PCR. All the necessary steps for theevaluation of transmission risk were considered in the protocol,including vector activity (inside and around the patient’s place ofresidence), and the efficacy of the control activities. The programmonitoring the cases of imported arbovirosis is integrated withina general procedure that monitors and treats monthly the areaswith recurrent proliferation of tiger mosquitoes (Bonnefoy et al.,2008; Montalvo et al., 2016).

Statistical AnalysisA descriptive study of qualitative and quantitative variables tocharacterize the study population was carried out. We computedthe frequency distributions of the qualitative variables, andcompared proportions using the χ

2-test, or the two-tailedFisher test when expected values were <5. Absolute frequencieswere calculated for categorical variables. Social, demographic,epidemiologic and clinical variables were compared accordingto the country of origin of the infected person (Spanish bornor foreign-born). The median and interquartilic range (IQR)for continuous variables was calculated and Spanish born andforeign born were compared using the U-Mann Whitney-test. Ap < 0.05 was considered statistically significant.

Ethical ConsiderationsThis study was approved by the Clinical Research EthicalCommittee of Parc Salut Mar (IMAS). In order to guaranteedata and registry confidentiality the regulation established bythe Organic Law of Personal Data Protection of Spain 15/1999and Royal Decree 994/1999 about security of computerized filesthat contain personal data was followed. All ethical principles forinvestigation in humans defined in the Declaration of Helsinki of1964 revised and updated by theWorldwide Medical Association(Fortaleza, Brazil 2013) were followed.

RESULTS

Epidemiological SurveillanceDuring the study period 118 cases where notified, 75 of which(63.6%) were laboratory confirmed. Figure 2 shows the monthlydistribution according to the place of residency (those who livedin the city or outside the city but diagnosed in Barcelona) ofthese 75 confirmed cases. Forty-four confirmed cases correspondto residents in the city, with an incidence of 2.74 cases per100,000 inhabitants. The median age was 35 (standard deviation11.8), 25 (57%) were women, and 19 (43%) men. They wereall imported cases, 21 (48%) among Spanish-born populationand 23 (52%) among immigrant population. No differencesregarding age and sex were observed (p = 0.2 and 0.57,respectively). The number of cases peaked in August, whenmost of the infections occurred among Spanish-born individuals(Figure 3).

When comparing the characteristics of the 44 confirmedcases between Spanish-born and foreign-born, it stands

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Millet et al. Zika Surveillance to Prevent Local Transmission

FIGURE 2 | Monthly distribution according to place of residence (living in the city or outside the city of Barcelona) of the first 75 confirmed cases of Zika virus infection

in Barcelona. Period: January–December 2016.

FIGURE 3 | Monthly distribution of the 44 imported cases of Zika virus in Barcelona according to country of birth (Spanish born or foreign born). Period:

January–December 2016.

out that the most frequently visited countries where theDominican Republic and Venezuela amongst the foreign-bornand Nicaragua, Colombia, Mexico, and Vietnam amongst theSpanish-born. Fifty-two percent of the cases didn’t reportbeing bitten by mosquitoes after arrival to Barcelona. Themost frequent clinical symptoms were rash (86%), fever(61%), and arthralgia (59%). No significant differences werefound between Spanish-born and foreign-born in relationto the clinical symptoms, visited country or detected bites(Table 1).

A total of 31 cases (70.5%) were arrived in Barcelonaduring the viremic period and 13 (30%) arrived during theincubation phase and became viremic in the city (Figure 4). Theproportion of viremic immigrants for 8–9 days in Barcelonawas higher than for the Spanish born (Table 1) (61% vs. 29%),p= 0.034.

The median time, for Spanish born, elapsed between thesymptoms onset and: (a) the first medical consultation date was 4days (IQR 2–5); (b) the notification date was 8 days (IQR 7–14);(c) the laboratory confirmation date was 9 days (IQR 6–12), and

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TABLE 1 | Comparison of the descriptive characteristics between Spanish born and foreign born for the 44 confirmed cases of Zika virus infection in the city of

Barcelona. January–November 2016.

Spanish-born N (%) Foreign-born N (%) Total N (%)

21 48% 23 52% 44

AGE (MEDIAN, SD) 33.5 10.7 36.5 12.7 35 11.8

SEX

Male 10 48% 9 39% 19 43%

Female 11 52% 14 61% 25 57%

HOSPITALIZATION

Yes 0 0% 0 0% 0 0%

No 21 100% 23 100% 44 100%

CLINICAL SYMPTOMS

Fever 14 67% 13 57% 27 61%

Arthralgia 11 52% 15 65% 26 59%

Rash 17 81% 21 91% 38 86%

Myalgia 6 29% 7 30% 13 30%

Cephalea 9 43% 9 39% 18 41%

Any other symptom 15 71% 12 52% 27 61%

COMPLICATIONS 0 0% 0 0% 0 0%

VISITED COUNTRY

Dominican Republic 2 10% 6 26% 8 18%

Nicaragua 6 29% 2 9% 8 18%

Colombia 3 14% 2 9% 5 11%

Mexico 3 14% 1 4% 4 9%

Venezuela 0 0% 4 17% 4 9%

Vietnam 3 14% 0 0% 3 7%

Honduras 1 5% 1 4% 2 5%

Bolivia 0 0% 2 9% 2 5%

Others 3 14% 5 22% 8 18%

DETECTED BITES

Yes 10 48% 11 48% 21 48%

No 11 52% 12 52% 23 52%

PREGNANCY 2 10% 2 14% 4 16%

DAYS OF RISK IN BARCELONA (VIREMIC PERIOD)

0 days 10 48% 3 13% 13 30%

1–7 days 5 24% 6 26% 11 25%

From 8 to 9 days 6 29% 14 61% 20 45%

PREVENTIVE RECOMMENDATIONS

Yes 21 100% 23 100% 44 100%

No 0 0% 0 0% 0 0%

(d) the implementation date of vector control activities was 9days (IQR 7–14). The median time in days for foreign born was 4(IQR 2–11), 11 (IQR 8–27), 20 (IQR 13–25), and 13 (IQR 11–28),respectively. No statistically significant differences were observedin these elapsed times between Spanish-born and foreign-bornpatients except for the lab confirmation days (p= 0.04).

Regarding the consequences of the ZIKV infection, nomortality was observed. Regarding morbidity, no GBS wasdetected. Among the 24 diagnosed women, 6 (25%) werepregnant. There was one case of microcephaly notified to PHABbut the pregnant woman lived outside Barcelona city.

Vector ControlAccording to the Surveillance protocol of arbovirosis inCatalonia, 34 entomological inspections related to ZIKV weredone between 1st April and 15th November. Nine of the 34 caseshad high mobility during the viremic period visiting differentareas of the city, increasing their exposure to tiger mosquitobites. Only 19 (43.2%) homes could be inspected, since thepersons could not be contacted or refused the inspection in theother 15 cases. The activity of A. albopictus was low, and wasonly detected in the public neighborhood of one of the cases(Figure 5).

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FIGURE 4 | Distribution of the first 44 confirmed cases of Zika virus infection according to the viremic days spent in the city of Barcelona.

There was a seasonal overlap between vector activity, andthe arrival of imported ZIKV cases with viremia (Figure 6).April, August, and September, are amongst the months of higheroverlap and consequently higher risk of local transmission ofZIKV.

To minimize the risk of transmission as much as possible, 122vector control interventions took place in public spaces and 12 inplaces of residence of ZIKV cases. Most of the interventions werelarvicide treatments in gutters and modifications of elementswith risk of hosting mosquito larvae, in most cases in privateproperty. In those cases in which tiger mosquito activity wasdetected or its presence was suspected, we used BG Sentineltraps for capturing mosquito females. Twelve traps were set, fourin public spaces, and eight in private property. Only one poolof mosquitoes was finally analyzed for ZIKV detection and theresult of the molecular analysis was negative.

DISCUSSION

The first year of epidemiological surveillance for ZIKV inthe city of Barcelona has allowed us to know very wellthe profile of ZIKV imported cases. The wide presence ofa vector that can transmit the virus (A. albopictus) and thepopulation movements increases the risk of introduction ofemerging arbovirusis like ZIKV. This fact requires surveillanceof the imported cases and tasks for vector control. This makesa good coordination between all the different actors, likeepidemiologists, clinicians, entomologists, and microbiologistsessential in order to avoid ZIKV from becoming a public

health problem that would also affect the tourism in thecity.

The imported ZIKV cases profile was that of a travelerthat after visiting endemic areas for ZIKV in Latin America,presents clinical symptoms such as fever, rash and arthralgia.No differences in sex, age, clinical symptoms, detected bites, orvisited country were observed among Spanish and foreign-bornpatients. Regarding the visited country, the predominant groupwas originally from the Dominican Republic, Venezuela, andNicaragua or Colombia. Other cities in Spain have also reported ahigh number of cases from Latin American countries. This is dueto the mobility of people between Latin America and Spain thattakes place for different reasons such as business, cooperation,tourism or visits to friends and relatives. It also responds to thehigh number of immigrants living in Spain especially since year2000, and that occasionally travel to their countries of origin(López De Lera, 1995; Díaz-Menéndez et al., 2016). In many ofthese countries ZIKV is endemic and its expansion is favored bythe wide presence and abundance of A. aegypti, main vector forZIKV and other arbovirosis.

ViremiaOver 70% of the diagnosed ZIKV cases in our city where viremic(PCR positive on serum) when they sought medical attention andover 36% (16/44) arrived in the city during incubation phase andspend all the viremic phase in our city. Usually tourists spend lesstime at their destinations than foreign born which also explainsthat Spanish-born patientsmore often present viremic phase afterarriving. For the foreign-born, the reason for traveling is to visitfriends and relatives and therefore normally they stay with their

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FIGURE 5 | Map of the city of Barcelona with the spatial location of the different arbovirosis cases and the results of the entomological inspections. Risk zones:

monthly surveillance and control vector zones. Citizen incidences: Notices regarding mosquito problems that citizens have indicated during the study period.

family and stay for a longer period of time. The cases that startedviremic phase after arriving to Spain represented the highest riskfor local transmission because of the longest duration of exposureto local vectors while still viremic. Additionally, an importantpercentage of patients reported mosquito bites after arrival, inmany cases overlapping with the viremic period.

The incubation period can vary between 2 and 14 daysafter infection and the viremic period can be 3–5 days longor longer (Falcao et al., 2016). Nevertheless, the viremic periodfor ZIKV has not been clearly established, especially in somevulnerable populations such as pregnant women (Suy et al.,2016). The viremic period in our city is estimated from thenatural history of the infection, 7 days starting from the dayof symptoms onset, not performing a repeated blood test tocheck the presence of the virus. Recently the CDC stated that theZIKV viremic period should be extended to 7 days, informationthat is of great relevance in vector control and autochthonoustransmission (Agència de Salut Pública de Catalunya, 2015).According to Figure 4 it can be deduced that if the viremic phasewas considered to be only 5 days, the total days for all casesin viremic period would be 116 days. However, if the viremic

phase is considered to be 9 days, the total days of viremic periodare 252 days, almost twice. Therefore, we believe that while theaccurate length of the viremic phase is not yet established, thebest option to maximize effects of epidemiological surveillanceand entomological precautions is to consider a viremic phase of9 days (Alejo-Cancho et al., 2016).

The duration of the viremic period has also importantimplications for the establishment of prevention measures.Entomologic inspections and tasks should occur as soon as asuspected case seeks medical advice, in order to reduce the riskof transmission during the viremic phase. Delays in the flowof information between medical and entomological staff maydramatically reduce the effectiveness in control, making themineffective if the information reaches the entomological staffwhen the viraemic period has finished.

It is important to highlight that 9 of 34 confirmed cases, inspite of the recommendations given by public health services orthe clinic, experienced great mobility during the viremic period.The fact that the clinical symptoms are frequently mild probablyhad an influence on this. These aspects together with the factthat most of the imported cases overlapped with periods of vector

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FIGURE 6 | Distribution of the Zika virus and Aedes albopictus vector activity (number of larval hatcheries detected on the public road) by month in the city of

Barcelona.

activity increased the risk of possible transmission. It is thereforevery important to recommend to patients a reduction in theirmobility during this period (Marrama-Rakotoarivony and Zeller,2012).

Zika Virus ConsequencesTo this date, nor deaths nor cases of SGB related to ZIKV havebeen reported in Barcelona. However, in a retrospective case-control study that took place after the ZIKV outbreak in FrenchPolynesia (2013–2014), 42 cases of SGB where identified whichwere also positive for DENV virus and ZIKV. Recently there hasbeen an increase in the SGB incidence in countries like Brazil,Colombia, El Salvador, and Suriname, but the exact cause is stillunknown.

Unfortunately, we found one microcephaly case out of 6babies born from ZIKV infected mothers. We had a high portionof pregnant woman due to the active screening of pregnantwoman that is being performed in Catalonia. Not only thedoctors but also pregnant woman that travel to a ZIKV endemicarea are aware about the risks of microcephaly in newbornand look for medical advice and follow up when they are backhome. This ongoing protocol in Catalonia perform not only anscreening and follow up of all pregnant woman exposed to therisk of ZIKV but also a long term follow-up of all newborn inorder to monitor the development of neurological abnormalities(Bocanegra et al., 2016). A prospective study in Brazil amongstpregnant women infected by ZIKV showed that 29% of thefetus suffered some type of abnormality during pregnancy,including microcephaly and intrauterine restricted development.Fetal abnormalities were detected by Doppler ultrasonographyin 12 of the 42 (29%) ZIKV positive women (Brasil et al., 2016;Mysorekar and Diamond, 2016; Reynolds et al., 2017). In light

of this scenario, we need to rapidly evaluate the risk to childdevelopment by this emerging disease that is spreading quicklyin many territories.

In the context of epidemiological surveillance it is importantnot to forget that ZIKV could be a sexual transmitted disease(STD). In the latest WHO update, 12 countries have reportedSTD of ZIKV, not only from man to woman, but also fromwoman to man, man to man (Deckard et al., 2016) and also couldbe transmitted by oral sex (World Health Organization, 2016).The exact duration of ZIKV in semen still remains unknown. Ina recent study, ZIKVRNAwas detected in semen 62 days after theinitiation of the symptoms ( D’Ortenzo et al., 2016), even thoughthe longest period in which ZIKV has been detected in semenis 188 days (Althaus and Low, 2016; Paz-Bailey et al., 2017).Other routes of transmission include transmission through blood(Barjas-Castro et al., 2016).

Reorganization of the Public HealthServicesAfter the diagnosis of the first imported cases of arbovirosis inour city, it was necessary to face up to the risk the presenceof A. albopictus represented to public health. For these reason,an epidemiologist and an entomologist were incorporated in theteam for surveillance and control of arbovirosis.

As a result, a protocol for the surveillance and controlof mosquito-borne arboviruses was established in Cataloniain 2014. It was written by an inter-institutional commissioncomprised of different experts (clinicians, microbiologists,entomologists, epidemiologists, etc., Agència de Salut Públicade Catalunya, 2015). The importance of surveillance and earlydetection was key therefore an interdisciplinary group formedby members of the Epidemiology Service and the Vector

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Control Service of the PHAB was created. Public health nurseswere trained to fully know the arboviruses and to adequatelycomplete the case reporting surveys. The structure (Figure 1)was also organized in coordination with the doctors of thehospitals specialized in Travel Medicine. This changes improvedcommunication systems, and accelerated the responses to risksituations that occurred with the arrival of viremic patients.A fundamental aspect was the communication and knowledgeof the protocol by all the involved agents. For this reason,PHAB organized training sessions for physicians were held inthe main hospitals. The aim was to increase awareness of theimportance of each agent involved in the process (Bonnefoyet al., 2008; Bueno, 2016). The information was disseminatedthrough the commission of arbovirosis of Catalonia and throughTV programs and news reports and at a round table that wasorganized at the 6th Emerging Diseases Congress in Barcelona(Camprubí, 2016).

In this first year of experience in ZIKV surveillance andcontrol, the delays observed among time elapsed between thesymptoms onset and first medical consultation, public healthnotification, laboratory confirmation, and implementation ofvector control activities, were remarkable. The first problemwas that the diagnostic delay in the first medical consultationand therefore the notification delay to the public health system.Therefore, the vector control activities were also initiated later,when patient was no longer viremic. So, from public health it isessential to educate patients to consult soon and also that doctorsnotify all suspected cases of ZIKV quickly.

Vector ControlTo this date no autochthonous ZIKV case has been detectedin Barcelona. However, they could have been occurred sincesome cases may goes under detected as happened in Croatia forDENV infection (Kurolt et al., 2013). There is a clear overlap ofthe peaks of imported cases and the vector’s activity (Figure 6).The analysis of the phenology in the vector appearance using aweighting of the risk factors, allows us to identify the monthsof April, August, and September (in general the months withhighest temperatures) as the most favorable for the mosquitospecies (Figure 3). In any case, the phenology of A. albopictusmatches the predictive models and the observations that havebeen carried out recently in different Mediterranean cities (Tranet al., 2013; Bueno, 2016).

Local transmission of ZIKV has been observed recently intemperate zones of USA such as Texas and Florida. Among the1,325 reported cases in 2016, 262 were due to local transmission,and 224 were in pregnant woman (16.9%). After governmentalefforts on vector control activities, no new ZIKV cases havebeen detected by local transmission in Florida. The example ofFlorida together with Barcelona, underlines the importance ofsurveillance and vector control activities and close monitoringto prevent ZIKV circulation (Department of Health Daily ZikaUpdate, 2017).

In light of this situation, efforts to intensify traveler’s advice,preventive measures and individual protection measures must bemaximized. We believe in paying special attention in providingresources to reduce mobility of the cases that are in viremic

period. This would help reduce contact with the vector anddecrease possible risk of transmission. A possible solution wouldbe to make home confinement obligatory. We are aware thatadherence to this recommendation could be low. In the case ofpeople that are working, an obligatory medical leave could beprovided in order to reduce mobility. Another area in whichimprovement could be made is in the inspection of privateproperties. These are confined areas where there is a great overlapbetween vector and viremic patient. In our case only 19 out of 34homes could be inspected, leaving a significant part of the riskassessment without completion.

In Barcelona, when vector activity was detected, eitherin larval phase or in adult phase, larvicide or adulticidecontrol actions took place within the framework of the basiccriteria of Plague Integrated Control (Bonnefoy et al., 2008).Adulticide spraying can pose several environmental problemsespecially in urban environments, since they are of largespectrum interventions that can affect non-target organisms.However, although the effect of adulticide treatments is wellknown to be short-lived, in certain epidemiological contexts awell technified adulticide application is needed to reduce themosquito population rapidly (Caputo et al., 2016). Consequently,it is a particularly interesting strategy in the framework ofimported cases of arbovirus in a concrete place where the vectorsare present in high densities. In any case, it is well knownthat a long temporal lapse between outbreak initiation andthe start of control tasks reduces effectiveness of adulticidesapplications significatively in terms of cost-benefits (Burattiniet al., 2008). Therefore, the best approach is to supply reactiveadulticides by preventive larvicides as a basis of the controlprogramme, since the treatment of potential breeding siteswith larvicides has a proven role in the reduction of adultpopulation at local scale in urban environments (Sochacki et al.,2016).

The activity of vectors detected in the entomologicalinspections of the ZIKV viremic cases in homes was low.However, additional inspections related to DENV cases detectedhigh activity for five cases during 2016. This fact highlightsthe need for coordination in the protocols for surveillanceand control of any arbovirosis in order to reduce the risk oftransmission (Lucientes-Curdi et al., 2014). Similar protocolshave been implemented in other countries such as Franceor Italy (Zammarchi et al., 2015; Maria et al., 2016) andrecently have also been approved at a national level inSpain (MSSSI, 2016). It is important that these programsfor surveillance and control integrate epidemiological issues(with an appropriate identification and follow up of cases andadequate prevention measures) as well as vector issues (vectorpresence and quantification) (Bonnefoy et al., 2008; Bueno,2016).

The use of new technology can help improve the systemsand knowledge of the territory (mosquito proliferation areasand detection of new competent vectors for transmission ofdiseases). Applications such as Mosquito Alert (http://www.mosquitoalert.com) can help improve the early detection systemand are of great help in managing this public health problem(Center for Ecological Research and Forestry Applications. Oltra

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et al., 2016).Mosquito Alert collaborated in locating unidentifiedbreeding sites in private property near confirmed cases ofZIKV.

A. albopictus may not be the only competent vector of ZIKVin Barcelona. Different studies have suggested the possible roleof Culex sp. as a ZIKV vector due to the occasional detection ofthe virus in wild populations (Huang et al., 2016). However, viralinfection experiments in the laboratory have not confirmed Culexas a competent vector (Amraoui et al., 2016; Boccolini et al., 2016;Huang et al., 2016).

Risk of Local Transmission and PreventiveMeasuresAutochthonous transmission of ZIKV can potentially occurin the Mediterranean countries since imported cases andcompetent vectors are present. Therefore, it is essential toestablish active case surveillance and prevention protocols toavoid local transmission. As previously stated, the most effectiveprevention measure to avoid local transmission is vector control.Additionally, it is important that healthcare professionals areinformed of the potential risk of ZIKV cases, since thiswill improve early case detection, surveillance procedures andtransmission control (Leona, 2016; Red Nacional de VigilancaEpidemiológica, 2016). Nevertheless, since a low percent of ZIKVare symptomatic, the majority of viremic travelers returningfrom endemic zones could be unidentified, and thereforepossible source of autochthonous outbreak. In this sense,reducing mosquito populations in the city may significantlyreduce the risk of autochthonous outbreak because limitingvector control activities to the neighborhood of symptomaticcases, may have limited impact if a significant fractionof ZIKV infections are asymptomatic during the viraemicphase.

In relation to the preventive measures, at an individual level,the imperative recommendation is that all people that travel toendemic areas should take precautions against mosquito bites(use of repellents, mosquito nets, long sleeve clothes) and onarrival should visit a medical center if any symptom develops(Agència de Salut Pública de Catalunya, 2015; Leona, 2016;MSSSI, 2016). At a community level, taking into considerationthat in the last few years migratory mobility and the increase ofindividual’s mobility has produced the introduction of emergingdiseases such as many arbovirosis, the recommendations wouldbe based on reducing the areas suitable for the reproductionof Aedes mosquitoes in around human inhabited areas. Thesemeasures would be based on the elimination or protection ofsmall containers that can accumulate water, and the applicationof measures of control in those water containers that cannot beprotected or eliminated. A frequent inspection of these spaces toavoid any mosquito breeding site would be necessary. Individualprotection measures should be taken in order to avoid bites,such as the use of long sleeves and long pants, and the use ofrepellents.

Pregnant women are a priority. The early detection ofthe cases and effects on fetus and newborns is essential. Inpregnant women, the diagnosis has to be confirmed and there

has to be a follow up of the pregnancy and the fetus. Menarriving from areas with local ZIKV transmission should havesafe sex during a minimum period of 6 months. Womenarrived from endemic areas should have safe sex during 8weeks (Red Nacional de Vigilanca Epidemiológica, 2016). Thepregnant women who have traveled to areas with local ZIKVtransmission or that have presented symptoms after the tripshould communicate this information during pre-natal visits.The time that a woman has to wait to get pregnant afterarriving from an affected area is 8 weeks from the time ofarrival or since the time of diagnosis (CDC, 2016; WHO,2016c).

All these preventive measures together with a goodmanagement of imported ZIKV cases and coordination amongepidemiologists, clinicians, entomologists, and microbiologistsare essential to prevent local transmission and therefore tolimit the extension of this emerging disease. The arbovirussurveillance program in Barcelona is an example of the needof a multidisciplinary approach in order to reduce the riskof introduction of the different arbovirosis, amongst them,ZIKV. The coordination between public health and pestcontrol agencies has contributed to the reduction of the risk ofautochthonous transmission.

ZIKA WORKING GROUP IN BARCELONA

Dolores Álamo-Junquera, Anna de Andrés, Ingrid Avellanés,Roser González, Pilar Gorrindo, Alexis Sentís, Pere Simón,Servicio de Epidemiología. Agencia de Salud Pública deBarcelona; Frederic Bartumeus, ICREA Movement EcologyLaboratory (CEAB-CSIC), Girona, Spain and CREAF (Center forEcological Research and Forestry Applications); Núria Busquets,IRTA, Centre de Recerca en Sanitat Animal, CReSA, IRTA-UAB, Barcelona, Spain; Izaskun Alejo, Joaquim Gascón, JoséMuñoz, Inés Oliveira, Ma Jesús Pinazo, Natalia Rodriguez,ISGlobal, Barcelona Ctr. Int. Health Res. (CRESIB), HospitalClínic-Universitat de Barcelona, Barcelona, Spain; CristinaBocanegra, Mateu Espasa, Israel Molina, Diana Pou, FernandoSalvador, Adrián Sánchez-Montalvà, Tomàs Pumarola, AriadnaRando, Nuria Serre, Antonio Soriano-Arandes, Begoña Treviño,Hospital Vall-d’Hebron-Drassanes, PROSICS, Barcelona, Spain;Mosquito Alert Community, Citizen scientists collaborating inthe platformMosquito Alert.

AUTHOR CONTRIBUTIONS

JM, TM, RB, EC, and JC conceived and designed the work, writethe first draft, revised critically and approved the final version.AR, AP analyzed and participated in the interpretation of thedata, revised critically and approved the final version. AR, AP,LF, LD, VP, MM, ES, and JF participated in the acquisition andinterpretation of the data, revised critically and approved the finalversion. Zika Working Group in Barcelona participated in theacquisition and interpretation of the data, revised critically andapproved the final draft. All authors agree to be accountable forall aspects of the work.

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ACKNOWLEDGMENTS

Our special gratitude to the Health Department in Catalonia, thePublic Health Agency of Catalonia and all the doctors, nursesand admin personnel in different hospitals and at primary carefor the good job performed together to fight against emergingarbovirosis. Special thanks to Silvia Brugueras for her greathelp with the translation. MJ, JG, NR, and JM receives fundsfrom 2014 SGR 26 grant from the Department d’Universitats,

Recerca i Societat de la Informacio de la Generalitat deCatalunya (AGAUR) and from RD12/0018/0010 grant, ISCIIIRETIC (MICINN, Spain). PROSICS authors are supported bythe 6th National Plan (PN) of Research + Development +

Innovation (I + D + I) 2008–2011, ISCIII-General DivisionNetworks and Cooperative Research Centres + FEDER funds +Collaborative Research Network on Tropical Diseases (RICET):RD12/0018/0020.

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Conflict of Interest Statement: The authors declare that the research wasconducted in the absence of any commercial or financial relationships that couldbe construed as a potential conflict of interest.

Copyright © 2017 Millet, Montalvo, Bueno-Marí, Romero-Tamarit, Prats-Uribe,

Fernández, Camprubí, del Baño, Peracho, Figuerola, Sulleiro, Martínez, Caylà

and Zika Working Group in Barcelona. This is an open-access article distributed

under the terms of the Creative Commons Attribution License (CC BY). The use,

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